The Flow Cytometry and Cellular Imaging Analysis Facility provides cellular analysis to investigators with peer-reviewed grants at M.D. Anderson Cancer Center. The facility encompasses 793 sq ft, and is directed by Dr. Michael Andreeff, who is assisted by one co-director, Dr. Walter Hittelman and 2 full-time and 1 part-time employee, Dr. Nicholas Terry, Wendy Schober, and Dr. Randall Evans. The facility develops and provides cutting-edge techniques in single-cell analysis. Cell phenotyping, proliferation, signaling, and apoptosis assays have been established and modified for multiparametric analysis. Immunophenotypic analysis was combined with assays of intracellular proteins related to apoptosis (Bcl-2, BcI-XL, BAG-1, p53, Rb, caspase activation, mitochondrial membrane potential and Fas), cell signaling (MAPK), proliferation (Ki67, cyclins, BrDU, proliferating cell nuclear antigen[PCNA], and DNA), and cell-division history (PKH-26). Quantitation of cellular antigens allows the determination of the antibody-binding capacity per cell. Very rare events and progenitor/stem cell subpopulations have been detected and isolated by 3-laser excitation/8-parameter fluorescence-activated cell sorting (FACS) for subsequent analysis by molecular cytogenetic and other molecular techniques. Acquisition of a high-speed sorter upgrade (Turbo Sort), a triple laser flow cytometer (B&D LSR) and a laser scanning cytometer (CompuCyte, Cambridge, MA) has upgraded the facility to provide state-of-the-art isolation and analysis, including quantitation of intracellular PKCalpha, Bax, Bcl-2, ERK, pERK, and XIAP. Laser confocal microscopy has been used extensively and was upgraded by acquisition of an Olympus FV-500 multi-user instrument that complements the Zeiss 510. High-impact studies in cancer prevention, growth factor signaling in breast cancer, apoptosis regulation in leukemias and multistep tumorigenesis all utilized confocal microscopy. The new Olympus allows live imaging at up to 4 fps. FISH has been combined with apoptosis assays to discriminate apoptosis in normal and malignant cells. The number, phenotype and proliferation of minimal residual disease cells could be determined at levels of 1 malignant cell in 30,000 normal cells. Methods for detection of transgene expression in cells are in place, using beta-galactosidase (beta-gal), nerve growth factor receptor (NGF-R), and enhanced green fluorescent protein (EGFP). This facility has 64 users from 18 programs; 70% of the users have peer-reviewed funding. The facility's use has increased from 26 to 55 investigators with peer-reviewed grants since the previous review that used the laser confocal microscopes for 1,298 hours and the FACS-LSC facility for 1,949 hours last year. The facility has continuously developed new methodologies in response to the evolving needs of its users.